Methods, kits, and antibodies for detecting parathyroid hormone

ABSTRACT

The present invention relates to novel methods and devices for detecting whole or non-fragmented parathyroid hormone (wPTH) in a biological sample. In particular, a novel monoclonal or polyclonal antibody or antibody fragment is used that is specific for a portion of the initial peptide sequence for wPTH which comprises a domain for adenylate cyclase activation, (amino acids 2 to 8), wherein at least four amino acids in this sequence are part of the reactive portion with the antibody. The present wPTH assay can differentiate between the complete 1 to 84 amino acid sequence form of PTH and a large but incomplete 7 to 84 amino acid sequence form of PTH that is measured by commercially available “intact” or I-PTH assays. Measurement of both the complete biologically active form of PTH and the large but biologically inactive form of PTH can lead to the misdiagnosis of the parathyroid function in patients.

TECHNICAL FIELD

The present invention relates to novel methods and devices for detectingwhole or non-fragmented parathyroid hormone (wPTH) in a biologicalsample. In particular, a novel monoclonal or polyclonal antibody orantibody fragment is used that is specific for a portion of the initialpeptide sequence for wPTH which comprises a domain for adenylate cyclaseactivation, (amino acids 2 to 8), wherein at least four amino acids arepart of the reactive portion with the antibody.

BACKGROUND ART

Calcium plays an indispensable role in cell permeability, the formationof bones and teeth, blood coagulation, transmission of nerve impulse,and normal muscle contraction. The concentration of calcium ions in theblood is, along with calcitrot and calcitonin, regulated mainly byparathyroid hormone (PTH). Although calcium intake and excretion mayvary, PTH serves through a feedback mechanism to maintain a steadyconcentration of calcium in cells and surrounding fluids. When serumcalcium lowers, the parathyroid glands secrete PTH, affecting therelease of stored calcium. When serum calcium increases, stored calciumrelease is retarded through lowered secretions of PTH.

The complete form of human PTH, (hPTH), is a unique 84 amino acidpeptide (SEQ ID NO. 2), as is shown in FIG. 1. Researchers have foundthat this peptide has an anabolic effect on bone that involves a domainfor protein kinase C activation (amino acid residues 28 to 34) as wellas a domain for adenylate cyclase activation (amino acid residues 1 to7). However, various catabolic forms of clipped or fragmented PTHpeptides also are found in circulation, most likely formed byintraglandular or peripheral metabolism. For example, whole PTH can becleaved between amino acids 34 and 35 to produce a (1-34) PTH N-terminalfragment and a (35-84) PTH C-terminal fragment. Likewise, clipping canoccur between either amino acids 36 and 37 or 37 and 38. Recently, alarge PTH fragment referred to as “non-(1-84) PTH” has been disclosedwhich is clipped closer to the N-terminal end of PTH. (See R. LePage elalia, “A non-(1-84) circulating parathyroid hormone (PTH) fragmentinterferes significantly with intact PTH commercial assay measurementsin uremic samples ” Clin Chem (1998); 44: 805-810.)

The clinical need for accurate measurement of PTH is well demonstrated.Serum PTH level is one of the most important index for patients with thefollowing diseases: familial hypocalciuric hypercalcemia; multipleendocrine neoplasia types I and II; osteoporosis; Paget's bone disease;primary hyperparathyroidism—caused by primary hyperplasia or adenoma ofthe parathyroid glands; pseudohypoparathyroidism, and renal failure,which can cause secondary hyperparathyroidism.

Determining circulating biologically active PTH levels in humans hasbeen challenging. One major problem is that PTH is found at low levels,normally 10 pg/mL to 40 pg/mL. Coupled with extremely low circulatinglevels is the problem of the heterogeneity of PTH and its manycirculating fragments. In many cases, immunoassays have facedsubstantial and significant interference from circulating PTH fragments.For example, some commercially available PTH kits have almost 100%cross-reactivity with the non-( 1-84) PTH fragment, (see the LePagearticle).

PTH immunoassays have varied over the years. One early approach is adouble antibody precipitation immunoassay found in U.S. Pat. No.4,369,138 to Arnold W. Lindall el alia. A first antibody has a highaffinity for a (65-84) PTH fragment. A radioactive labeled (65-84) PTHpeptide is added to the sample with the first antibody to compete forthe unlabeled peptide. A second antibody is added which binds to anyfirst antibody and radioactive labeled PTH fragment complex, therebyforming a precipitate. Both precipitate and supernatant can be measuredfor radioactive activity, and PTH levels can be calculated therefrom.

In an effort to overcome PTH fragment interference, immunoradiometrictwo-site assays for intact PTH (I-PTH) have been introduced, such asAllegro® Intact PTH assay by the Nichols Institute of San JuanCapistrano, Calif. In one version, a capture antibody specifically bindsto the C-terminal portion of hPTH while a labeled antibody specificallybinds to the N-terminal portion of the captured hPTH. In another, twomonoclonal antibodies were used, both of which attached to theN-terminal portion of hPTH. (For the purposes of the present invention,the complete form of human PTH is referred to as “whole PTR” or “wPTH”as distinguished from “intact PTH” or “I-PTH” which can include not onlywPTH, but also a large PTH fragment cleaved about amino acids 5 to 8.)Unfortunately, these assays have problems in that they measure but donot discriminate between w-PTH and I-PTH. This inability comes to thefore in hyperparathyroid patients and renal failure patients who havesignificant endogenous concentrations of large, non-whole PTH fragments.

Recently, researchers have made a specific binding assay directed to thelarge N-terminal PTH fragments. (See. Gao, Ping et alia“Immunochemicalluminometric assay with two monoclonal antibodies againstthe N-terminal sequence of human parathyroid hormone”, Clinica ChimicaActa 245 (1996) 39-59.) This immunochemiluminometric assay uses twomonoclonal antibodies to detect N-terminal (1-34) PTH fragments but notmid-portion PTH fragments or C-terminal PTH fragments. A key factor inthe design of these assays is to eliminate any reaction with C-terminalPTH fragments.

DISCLOSURE OF THE INVENTION

The present invention relates to a method for detecting wPTH in abiological sample without detecting the non (1-84) large PTH fragmentcomponent of I-PTH, and in particular to a substantially pure monoclonalor polyclonal antibody or antibody fragment specific for the initialsequence for wPTH which comprises a domain for adenylate cyclaseactivation, VAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO. 3), wherein at leastfour amino acids in this sequence are part of the antibody reactiveportion of the peptide. The method for measuring the amount of wPTH in asample such as serum, plasma, or blood comprises four general stepswhich can vary depending upon whether one uses a first antibody orantibody fragment specific for the PTH peptideVAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO. 3), wherein at least four aminoacids are part of the antibody reactive portion of the peptide either asa signal antibody or a capture antibody in conventional immunoassayformats. Used either as a signal antibody or as a capture antibody,enough antibody is added to bind all w-PTH present. Next, one allows thefirst antibody to bind to any wPTH present, thereby forming a complex. Aspecific binding label comprised of a second antibody and a conventionalimmunoassay label, such as chemiluminescent agents, colorimetric agents,energy transfer agents, enzymes, fluorescent agents, and radioisotopes,is used to label the complex, preferably at the N-terminal end of wPTH,and can be added either substantially simultaneously with the firstantibody or subsequent thereto. Finally, one uses conventionaltechniques to measure the amount of labeled complex, and therebycalculate wPTH levels in the sample. If used as a signal antibody, thenthe first.

One can also use the present invention to detect the amount ofN-terminal PTH fragment present having a complete and functional aminoacid sequence from 1 to at least 34. Some researchers are usingsynthetic N-terminal PTH peptides as a therapeutic treatment to stopbone loss and actually encourage an increase in bone mass. Thesepeptides range from amino acids 1 to at least 34 and on up to 38. Thepresent assay can detect the amount and duration of such syntheticpeptides in circulation in a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view of wPTH.

FIG. 2 is a diagrammatic view of a wPTH assay using the present antibodyas a tracer element.

FIG. 3 is a diagrammatic view of a wPTH assay using the present antibodyas a capture element.

FIG. 4 is a graph showing a standard curve for a wPTH assay.

FIGS. 5 are diagrammatic views showing binding of whole (1-84) PTHcompared with interference from non (1-84) PTH fragments (e.g., (7-84)PTH (SEQ ID NO:6)) in conventional I-PTH assays.

FIGS. 6A and 6B are diagrammatic views showing binding of whole (1-84)PTH compared with interference from non (1-84) PTH fragments (e.g.,(7-84) PTH (SEQ ID NO:6)) in conventional I-PTH assays.

FIG. 6 is a diagrammatic view showing interference from non (1-84) PTHfragments in conventional I-PTH assays.

FIG. 7 is a graph comparing a conventional I-PTH assay with the presentwPTH assay for patients with chronic uremia.

FIG. 8 is a graph showing the distribution of values for healthy normalpersons, patients with primary hyperparathyroidism, and patients withchronic uremia.

BEST MODES FOR CARRYING OUT THE INVENTION Whole PTH Immunoassay

A preferred embodiment of the present invention is an immunoradiometricassay (IRMA), often referred to as a sandwich assay, as shown FIGS. 2and 3. Elements employed in such an assay (10) include a captureantibody (12) attached to a solid support (14) and a signal antibody(16) having a label (18), attached thereto (20). Typically, one selectsa capture antibody that is specific for C-terminal PTH fragments (22),while the label antibody is specific for the initial wPTH peptidesequence which comprises a domain for adenylate cyclase activation (24),as shown in FIG. 2. However, one could reverse the specificity of theseantibodies, as is shown in FIG. 3.

Alternatively, one could create an immunoassay in which wPTH is eitherprecipitated from solution or otherwise differentiated in a solution, asin a conventional precipitating assays or turbidometric assays. Forexample, one can use using at least three antibodies to form aprecipitating mass. In addition to the initial wPTH sequence antibodyand a C-terminal antibody, one can use at least a third antibody whichattaches to the mid portion of PTH. The combined mass of wPTH and the atleast three antibodies would form a labeled precipitating mass which canbe measured by conventional techniques. Another method would be tocouple the initial wPTH sequence antibody to colloidal solid supports,such as latex particles.

More specifically, one can create a signal antibody by iodinating 50micrograms of affinity purified goat anti-(1-8) PTH antibody(Scantibodies Laboratory, Inc., Santee Calif., U.S.A.) by oxidation withchloramine T, incubation for 25 seconds at room temperature with 1millicurie of 125-I radioisotope and reduction with sodiummetabisulfate. Unincorporated 125-I radioisotope is separated from the125-1-Goat anti-(1-8) PTH signal antibody by, passing the iodinationmixture over a PD-10 desalting column (Pharmacia, Uppsala, Sweden) andfollowing the manufacturers instructions. The fractions collected fromthe desalting column are measured in a gamma counter and those fractionsrepresenting the 125-1-Goat anti-(1-8) PTH antibody are pooled anddiluted to approximately 300,000 DPM (disintegrations per minute) per100 microliters. This solution is the tracer solution to be used in thewhole PTH IRMA.

A capture antibody can be created by attaching affinity purified goatanti PTH 39-84 antibody, (Scantibodies Laboratory, Inc., Santee, Calif.,U.S.A.), to 12×75 mm polystyrene tubes (Nunc, Denmark) by means ofpassive absorption techniques which are known to those of skill in theart. The tubes are emptied and dried, creating solid phase antibodycoated tubes.

To conduct an assay of a sample, 200 microliter samples of human serumare added to the solid phase antibody coated tubes. To each tube isadded 100 microliters of the tracer solution (labeled goat anti-(1-8)PTH signal antibody). The tubes are incubated at room temperature withshaking for 170 rpm for 20-22 hours. During this time the immunochemicalreaction of forming the sandwich of (solid phase goat anti-(39-84) PTHantibody)—(whole PTH)—(125-1-goat anti-(1-8) PTH antibody) takes place.Following this incubation, the test tubes are washed with distilledwater. Radioactivity on the solid phase, which amount corresponds to thequantity of wPTH present, is measured using a gamma counter. Theradioactivity data for the samples is processed by conventional analysiswith use of the results from standards and controls and a computersoftware in order that the concentration of whole PTH in the samples maybe ascertained. FIG. 4 shows a standard curve for such an assay.

Initial Whole PTH Sequence Peptide

In order to make the signal antibody in the above assay, first one makesa synthetic PTH peptide corresponding either to hPTH(Ser-Val-Ser-Glu-lie-Gln-Leu-Met), SEQ ID NO:4, rat PTH(Ala-Val-Ser-Glu-lie-Gln-Leu-Met), SEQ ID NO:5, or at least four aminoacids in the common sequence, absent the first amino acid. The selectedpeptide can play two roles in making an assay, first as a specificantigenic source for creating a polyclonal antibody or monoclonalantibody source for signal antibody or capture antibody, and second aspart of an affinity purification means for isolating the desired signalantibody or capture antibody.

Briefly, such a peptide can be synthesized on an Applied Biosystems,Inc. (Foster City, Calif., U.S.A.) Model 431 automated peptidesynthesizer employing Fmoc (9-fluoronylmethoxycarbonyl) as thealpha-amino protecting group. All amino acids and solvents are fromApplied Biosystems and are of synthesis grade. Following synthesis, thepeptide is cleaved from the resin, and side chains are de-blocked, usinga cleavage cocktail containing 6.67% phenol, 4.4% (v/v) thioanisole and8.8% ethanedithiol in trifluoroacetic acid (TFA). The cleaved peptide isprecipitated and washed several times in cold diethyl ether. It is thendissolved in water and lyophilized. The crude peptide is subjected toamino acid analysis (Waters PICO-TAG System, Boston, Mass., U.S.A.) andreversed-phase HPLC using a VYDAC (TM) C8 column with 0.1% TFA in waterand 99.9% acetonitrile in 0.1% TFA as the mobile buffers. The presenceof a single major peak along with the appropriate amino acid compositionis taken as evidence that the peptide is suitable for further use.

The resulting peptide is then attached to cross linked agarose beads(Sepharose 4B from Pharmacia, Uppsala, Sweden) according to instructionsfrom the manufacturer. Armed with the initial peptide sequence on abead, one can affinity purify a polyclonal antibody serum source toisolate the initial sequence antibody for the wPTH immunoassay.

Initial Sequence Whole PTH Antibody

To create an affinity-purified anti-(1-8) PTH antibody, one first uses aselected initial PTH sequence peptide as described above as part of animmunogen for injection into a goat. The peptide can be used either byitself as an injectible immunogen, incorporated into a non PTH peptidehaving a molecular weight, typically, of between about 5000 and10,000,000, or as part of the wPTH complete sequence. The immunogen ismixed with an equal volume of Freunds complete adjuvant which is amixture of light mineral oil and inactivated mycobactcrium tuberculosisbacilli. The resulting mixture is homogenized to produce an aqueous/oilemulsion which is injected into the animal (typically a goat) for theprimary immunization. The immunogen dose is approximately 50-400micrograms. The goats are injected monthly with the same dose ofimmunogen complex except no mycobacterium tuberculosis bacilli is usedin these subsequent injections. The goats are bled monthly,approximately three months after the primary immunization. The serum (orantiserum) is derived from each bleeding by separating the red bloodcells from the blood by centrifugation and removing the antiserum whichis rich in (1-8) PTH antibodies.

To purify the antiserum for the desired (1-8) PTH antibody, one packs aseparation column with the initial PTH sequence peptide bound beadsdescribed above, washes the column and equilibrates it with 0.01 Mphosphate buffered saline (PBS). The antiserum is loaded onto the columnand washed with 0.01 M PBS in order to remove antibodies without the(1-8) PTH specificity. The bound specific goat anti-(1-8) PTH polyclonalantibody is eluted from the solid phase PTH 1-8 in the column by passingan elution solution of 0.1 M glycine hydrochloride buffer, pH 2.5through the column. The eluted polyclonal antibody is neutralized afterit leaves the column with either the addition of I M phosphate buffer,pH 7.5 or by a buffer exchange with 0.01 M PBS, as is known to those ofskill in the art. The polyclonal antibody is stored at 2-8 degreescentigrade.

Comparison Between Whole PTH and Intact PTH Assays

The present IRMA assay was compared to a conventional I-PTH immunoassay,the Allegro Nichols Intact PTH assay, (which is commercially availableand made by Nichols Institute Diagnostics of San Juan Capistrano,Calif., U.S.A.), in both PTH normal patients and those suffering fromchronic uremia.

FIG. 5 shows the results for 34 normal human serum samples from healthysubjects which were assayed both by the present wPTH IRMA and the aboveI-PTH assay. In every case, the level of wPTH detected by the IRMA islower than that reported by the I-PTH assay, demonstrating the abilityof the present IRMA to avoid detecting the interfering large, non (1-84)PTH fragments detected by the I-PTH assay. FIGS. 6A and 6B illustratehow such interference can occur. An N-terminal PTH specific signalantibody which is not specific to the initial PTH peptide sequence, asin the present invention, can detect not only wPTH (as in FIG. 6A), butalso can detect large, non (1-84) PTH fragments (as in FIG. 6B).

A comparison of assay results for 161 chronic uremia patients is shownin FIG. 7. Serum samples from these patients were measured using thewPTH IRMA and the above I-PTH assay. In every case the wPTH levels arelower than I-PTH values.

Clinical Use

The present wPTH assay has been used in a clinical setting involving 245persons. The group included 32 persons having normal healthy parathyroidglands, 52 patients with pathologically confirmed primaryhyperparathyroidism (1⁰ HPT), and 161 patients with chronic uremia whoare undergoing dialysis on a continuous basis. FIG. 8 illustratespatient differentiating results using the wPTH assay. A person havingsubstantially normal parathyroid hormone function can be differentiatedfrom one having hyperparathyroidism by measuring whole parathyroidhormone levels. Moreover, chronic uremia patients can be differentiatedinto two groups by measuring whole parathyroid hormone levels in theperson and comparing them to normal values, namely, those havingsubstantially normal active parathyroid hormone levels, and those havinghyperparathyroidism (or secondary hyperparathyroidism).

The ordinarily skilled artisan can appreciate that the present inventioncan incorporate any number of the preferred features described above.

All publications or unpublished patent applications mentioned herein arehereby incorporated by reference thereto.

Other embodiments of the present invention are not presented here whichare obvious to those of ordinary skill in the art, now or during theterm of any patent issuing from this patent specification, and thus, arewithin the spirit and scope of the present invention.

We claim:                    #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 6 <210> SEQ ID NO 1 <211> LENGTH: 7<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1Ser Val Ser Glu Ile Gln Leu 1               5 <210> SEQ ID NO 2<211> LENGTH: 84 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 2 Ser Val Ser Glu Ile Gln Leu Met His Asn L#eu Gly Lys His Leu Asn  1               5   #                10  #                15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys L#ys Leu Gln Asp Val His             20       #            25      #            30 Asn Phe Val Ala Leu Gly Ala Pro Leu Ala P#ro Arg Asp Ala Gly Ser         35           #        40          #        45 Gln Arg Pro Arg Lys Lys Glu Asp Asn Val L#eu Val Glu Ser His Glu     50               #    55              #    60 Lys Ser Leu Gly Glu Ala Asn Lys Ala Asp V#al Asn Val Leu Thr Lys 65                   #70                  #75                   #80 Ala Lys Ser Gln <210> SEQ ID NO 3<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 3 Val Ser Glu Ile Gln Leu Met 1               5<210> SEQ ID NO 4 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Rat<400> SEQUENCE: 4 Ser Val Ser Glu Ile Gln Leu Met 1               5<210> SEQ ID NO 5 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Rat<400> SEQUENCE: 5 Ala Val Ser Glu Ile Gln Leu Met 1               5<210> SEQ ID NO 6 <211> LENGTH: 78 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 6Leu Met His Asn Leu Gly Lys His Leu Asn S #er Met Glu Arg Val Glu 1               5   #                10   #                15Trp Leu Arg Lys Lys Leu Gln Asp Val His A #sn Phe Val Ala Leu Gly            20       #            25       #            30Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser G #ln Arg Pro Arg Lys Lys        35           #        40           #        45Glu Asp Asn Val Leu Val Glu Ser His Glu L #ys Ser Leu Gly Glu Ala    50               #    55               #    60Asn Lys Ala Asp Val Asn Val Leu Thr Lys A #la Lys Ser Gln65                   #70                   #75


1. A substantially pure antibody or antibody fragment specific for aninitial peptide sequence of whole parathyroid hormone wherein saidinitial peptide sequence consists of VAL-SER-GLU-ILE-GLN-LEU-MET (SEQ IDNO:3), and wherein at least four amino acids in said initial peptidesequence are part of a reactive portion with said antibody.
 2. Theantibody of claim 1, which is a monoclonal antibody.
 3. The antibody ofclaim 1, which is a polyclonal antibody.
 4. The antibody of claim 1,which antibody specifically binds to a parathyroid hormone epitopeselected from the group consisting of amino acid residues 2-5, 3-6, 4-7,5-8, 2-6, 3-7 and 4-8 of human parathyroid hormone (SEQ ID NO:2).
 5. Amethod for measuring an amount of whole parathyroid hormone in a samplecomprising: a) adding to a sample a labeled antibody or antibodyfragment specific for an initial peptide sequence of whole parathyroidhormone wherein said initial peptide sequence consists ofVAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO:3), and wherein at least fouramino acids in said initial peptide sequence are part of a reactiveportion to said labeled antibody; b) allowing said labeled antibody tobind to whole parathyroid hormone present, thereby forming a complex;and c) measuring the amount of said labeled complex to measure theamount of whole parathyroid hormone in said sample while not detectingan interfering non-(1-84) parathyroid hormone fragment.
 6. The method ofclaim 5, wherein the labeled anti-parathyroid hormone antibody orantibody fragment is a monoclonal antibody.
 7. The method of claim 5,wherein the labeled anti-parathyroid hormone antibody or antibodyfragment is a polyclonal antibody.
 8. The method of claim 5, wherein asecond antibody is added which is bound to a solid support andspecifically binds to a portion of whole parathyroid hormone other thanthe initial peptide sequence which binds to the labeled antibody.
 9. Themethod of claim 8, wherein the solid support is selected from the groupconsisting of a protein binding surface, colloidal metal particles, ironoxide particles, latex particles and polymeric beads.
 10. The method ofclaim 9, wherein the complex precipitates from solution.
 11. The methodof claim 5, wherein the label of the labeled antibody is selected fromthe group consisting of a chemiluminescent agent, a colorimetric agent,an energy transfer agent, an enzyme, a fluorescent agent and aradioisotope.
 12. The method of claim 5, wherein the labeled antibodyspecifically binds to a parathyroid hormone epitope selected from thegroup consisting of amino acid residues 2-5, 3-6, 4-7, 5-8, 2-6, 3-7 and4-8 of human parathyroid hormone (SEQ ID NO:2).
 13. A method formeasuring an amount of whole parathyroid hormone in a sample comprising:a) adding to a sample a first antibody or antibody fragment specific foran initial peptide sequence of whole parathyroid hormone wherein saidinitial peptide sequence consists of VAL-SER-GLU-ILE-GLN-LEU-MET (SEQ IDNO:3), and wherein at least four amino acids in said initial peptidesequence are part of a reactive portion to said first antibody; b)allowing said first antibody to bind to whole parathyroid hormonepresent, thereby forming a complex; c) labeling said complex by adding asecond labeled antibody that specifically binds to a portion of wholeparathyroid hormone other than said initial peptide sequence that bindsto said first antibody to form a labeled complex; and d) measuring theamount of said labeled complex to measure the amount of wholeparathyroid hormone in said sample while not detecting an interferingnon-(1-84) parathyroid hormone fragment.
 14. The method of claim 13,wherein the second labeled antibody is added sequentially orsimultaneously with the first antibody.
 15. The method of claim 13,wherein the first antibody is bound to a solid support.
 16. The methodof claim 13, further comprising binding a third antibody to an epitopeleft open after the whole parathyroid hormone binds to the firstantibody and the second antibody, thereby forming a precipitating mass.17. The method of claim 13, wherein the first antibody specificallybinds to a parathyroid hormone epitope selected from the groupconsisting of amino acid residues 2-5, 3-6, 4-7, 5-8, 2-6, 3-7 and 4-8of human parathyroid hormone (SEQ ID NO:2).
 18. A method for measuringwhole parathyroid hormone by a precipitating or turbidometricimmunoassay comprising: a) adding to a sample an antibody or antibodyfragment specific for an initial peptide sequence of whole parathyroidhormone wherein said initial peptide sequence consists ofVAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO:3), and wherein at least fouramino acids in said initial peptide sequence are part of an antibodyreactive portion of said peptide, said antibody being attached to acolloidal particle or moiety which can be used to detect a signalchange; b) allowing said antibody to bind to whole parathyroid hormonepresent, thereby forming a complex; and c) measuring change in saidsignal due to the formation of said complex to measure whole parathyroidhormone in said sample while not detecting an interfering non-(1-84).parathyroid hormone fragment.
 19. The method of claim 18, wherein thefirst antibody specifically binds to a parathyroid hormone epitopeselected from the group consisting of amino acid residues 2-5, 3-6, 4-7,5-8, 2-6, 3-7 and 4-8 of human parathyroid hormone (SEQ ID NO:2).
 20. Akit for assaying for whole parathyroid hormone comprising: a) asubstantially pure antibody or antibody fragment specific for an initialpeptide sequence of whole parathyroid hormone wherein said initialpeptide sequence consists of VAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO:3),and wherein at least four amino acids in said initial peptide sequenceare part of a reactive portion with said antibody; and b) a labelingcomponent that binds to whole parathyroid hormone, but not to saidparathyroid hormone initial peptide sequence VAL-SER-GLU-ILE-GLN-LEU-MET(SEQ ID NO:3).
 21. The kit of claim 20, wherein the substantially pureantibody or antibody fragment specifically binds to a parathyroidhormone epitope selected from the group consisting of amino acidresidues 2-5, 3-6, 4-7, 5-8, 2-6, 3-7 and 4-8 of human parathyroidhormone (SEQ ID NO:2).
 22. A kit for assaying for whole parathyroidhormone comprising: a) a substantially pure antibody or antibodyfragment specific for an initial peptide sequence of whole parathyroidhormone wherein said initial peptide sequence consists ofVAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO:3), and wherein at least fouramino acids in said initial peptide sequence are part of a reactiveportion with said antibody; and b) a second antibody bound to a solidsupport and said second antibody is specific for a portion of wholeparathyroid hormone that does not include the domain for adenylatecyclase activation.
 23. The kit of claim 22, further comprising a thirdantibody specific for an epitope left open after the whole parathyroidhormone binds to the first and the second antibodies, thereby forming aprecipitating mass.
 24. The kit of claim 22, wherein the substantiallypure antibody or antibody fragment specifically binds to a parathyroidhormone epitope selected from the group consisting of amino acidresidues 2-5, 3-6, 4-7, 5-8, 2-6, 3-7 and 4-8 of human parathyroidhormone (SEQ ID NO:2).
 25. A method for measuring an amount of afunctional N-terminal parathyroid hormone fragment and whole parathyroidhormone in a sample comprising: a) adding to a sample a first antibodyor antibody fragment specific for an initial peptide sequence of wholeparathyroid hormone wherein said initial peptide sequence consists ofVAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO:3), and wherein at least fouramino acids in said initial peptide sequence are part of a reactiveportion with said first antibody; b) adding to said sample a secondantibody or antibody fragment specific for a peptide comprising aminoacid sequence 28 to 34 of human parathyroid hormone (SEQ ID NO:2), whichcomprises a domain for protein kinase C activation, wherein at leastfour amino acids in said peptide sequence are a reactive portion withsaid second antibody; c) allowing said first antibody and secondantibody, wherein at least one of which is labeled, to bind toN-terminal parathyroid hormone fragment or whole parathyroid hormonepresent in said sample, thereby forming a labeled complex; and d)measuring the amount of said labeled complex to measure the amount ofsaid functional N-terminal parathyroid hormone fragment and wholeparathyroid hormone in said sample while not detecting an interferingnon-(1-84) parathyroid hormone fragment.
 26. The method of claim 25,wherein the first antibody or antibody fragment specifically binds to aparathyroid hormone epitope selected from the group consisting of aminoacid residues 2-5, 3-6, 4-7, 5-8, 2-6, 3-7 and 4-8 of human parathyroidhormone (SEQ ID NO:2).
 27. A method for differentiating between a personhaving substantially normal parathyroid hormone function and havinghyperparathyroidism comprising: a) obtaining a sample from a person tobe tested; b) contacting said sample with a substantially pure antibodyor antibody fragment specific for an initial peptide sequence of wholeparathyroid hormone wherein said initial peptide sequence consists ofVAL-SER-GLU-ILE-GLN-LEU-MET (SEQ ID NO:3), and wherein at least fouramino acids in said initial peptide sequence are part of a reactiveportion with said antibody; and c) assessing binding between saidsubstantially pure antibody or antibody fragment and whole parathyroidhormone, if present in said sample, to measure whole parathyroid hormonelevel in said person, while not detecting an interfering non-(1-84)parathyroid hormone fragment, and to determine if said person hassubstantially normal parathyroid hormone function or hashyperparathyroidism.
 28. The method of claim 27, wherein thehyperparathyroidism is primary hyperparathyroidism.
 29. The method ofclaim 27, wherein the hyperparathyroidism is secondaryhyperparathyroidism.
 30. The method of claim 27, wherein thehyperparathyroidism is caused by chronic renal failure.
 31. The methodof claim 27, wherein the substantially pure antibody or antibodyfragment specifically binds to a parathyroid hormone epitope selectedfrom the group consisting of amino acid residues 2-5, 3-6, 4-7, 5-8,2-6, 3-7 and 4-8 of human parathyroid hormone ID NO:2).
 32. The methodof claim 27, further comprising comparing the whole parathyroid hormonelevel determined in said tested person to a whole parathyroid hormonelevel determined in a normal person without hyperparathyroidism.
 33. Themethod of claim 5, wherein the sample is a rat or human sample.
 34. Themethod of claim 14, wherein the sample is a rat or human sample.
 35. Themethod of claim 18, wherein the sample is a rat or human sample.
 36. Themethod of claim 27, wherein the sample is a rat or human sample.